Anticodon Loop Research Articles

Structural evolution of Glycyl-tRNA synthetases alpha subunit and its implication in the initial organization of the decoding system

The origin and evolution of the genetic code is a fundamental challenge in modern biology. At the center of this problem is the correct interaction between amino acids and tRNAs. Aminoacyl-tRNA synthetase is the enzyme responsible for the correct binding between amino acids and tRNAs. Among the 20 canonical amino acid, glycine was the most abundant in prebiotic condition and it must have been one of the first to be incorporated into the genetic code. In this work, we derive the ancestral sequence of Glycyl-tRNA synthetase (GlyRS) and predict its 3D-structure. We show, via molecular docking experiments, the capacity of ancestral GlyRS to bind the tRNA anticodon stem loop, cofactors and substrates. These bindings exhibit high affinity and specificity. We propose that the primordial function of these interactions was to stabilize both compounds to make possible the catalysis. In this context, the anticodon stem loop did contribute to the encoding system and just with the emergence of the mRNA it was co-opted for codification. Thus, we present a model for the origin of the genetic code in which the operational and the anticodon codes did not evolve independently.

A rationale for tRNA modification circuits in the anticodon loop.

The numerous post-transcriptional modifications of tRNA play a crucial role in tRNA function. While most modifications are introduced to tRNA independently, several sets of modifications are found to be interconnected such that the presence of one set of modifications drives the formation of another modification. The vast majority of these modification circuits are found in the anticodon loop (ACL) region where the largest variety and highest density of modifications occur compared to the other parts of the tRNA and where there is relatively limited sequence and structural information. We speculate here that the modification circuits in the ACL region arise to enhance enzyme modification specificity by direct or indirect use of the first modification in the circuit as an additional recognition element for the second modification. We also describe the five well-studied modification circuits in the ACL, and outline possible mechanisms by which they may act. The prevalence of these modification circuits in the ACL and the phylogenetic conservation of some of them suggest that a number of other modification circuits will be found in this region in different organisms.

Tautomeric G•U pairs within the molecular ribosomal grip and fidelity of decoding in bacteria.

We report new crystallographic structures of Thermus thermophilus ribosomes complexed with long mRNAs and native Escherichia coli tRNAs. They complete the full set of combinations of Watson–Crick G•C and miscoding G•U pairs at the first two positions of the codon–anticodon duplex in ribosome functional complexes. Within the tight decoding center, miscoding G•U pairs occur, in all combinations, with a non-wobble geometry structurally indistinguishable from classical coding Watson–Crick pairs at the same first two positions. The contacts with the ribosomal grip surrounding the decoding center are all quasi-identical, except in the crowded environment of the amino group of a guanosine at the second position; in which case a G in the codons may be preferred. In vivo experimental data show that the translational errors due to miscoding by G•U pairs at the first two positions are the most frequently encountered ones, especially at the second position and with a G on the codon. Such preferred miscodings involve a switch from an A-U to a G•U pair in the tRNA/mRNA complex and very rarely from a G = C to a G•U pair. It is concluded that the frequencies of such occurrences are only weakly affected by the codon/anticodon structures but depend mainly on the stability and lifetime of the complex, the modifications present in the anticodon loop, especially those at positions 34 and 37, in addition to the relative concentration of cognate/near-cognate tRNA species present in the cellular tRNA pool.

Oligonucleotide-mediated tRNA sequestration enables one-pot sense codon reassignment in vitro.

Sense codon reassignment to unnatural amino acids (uAAs) represents a powerful approach for introducing novel properties into polypeptides. The main obstacle to this approach is competition between the native isoacceptor tRNA(s) and orthogonal tRNA(s) for the reassigned codon. While several chromatographic and enzymatic procedures for selective deactivation of tRNA isoacceptors in cell-free translation systems exist, they are complex and not scalable. We designed a set of tRNA antisense oligonucleotides composed of either deoxy-, ribo- or 2′-O-methyl ribonucleotides and tested their ability to efficiently complex tRNAs of choice. Methylated oligonucleotides targeting sequence between the anticodon and variable loop of tRNASerGCU displayed subnanomolar binding affinity with slow dissociation kinetics. Such oligonucleotides efficiently and selectively sequestered native tRNASerGCU directly in translation-competent Escherichia coli S30 lysate, thereby, abrogating its translational activity and liberating the AGU/AGC codons. Expression of eGFP protein from the template harboring a single reassignable AGU codon in tRNASerGCU-depleted E. coli lysate allowed its homogeneous modification with n-propargyl-l-lysine or p-azido-l-phenylalanine. The strategy developed here is generic, as demonstrated by sequestration of tRNAArgCCU isoacceptor in E. coli translation system. Furthermore, this method is likely to be species-independent and was successfully applied to the eukaryotic Leishmania tarentolae in vitro translation system. This approach represents a new direction in genetic code reassignment with numerous practical applications.

Controlling the Replication of a Genomically Recoded HIV-1 with a Functional Quadruplet Codon in Mammalian Cells.

Large efforts have been devoted to genetic code engineering in the past decade, aiming for unnatural amino acid mutagenesis. Recently, an increasing number of studies were reported to employ quadruplet codons to encode unnatural amino acids. We and others have demonstrated that the quadruplet decoding efficiency could be significantly enhanced by an extensive engineering of tRNAs bearing an extra nucleotide in their anticodon loops. In this work, we report the identification of tRNA mutants derived from directed evolution to efficiently decode a UAGA quadruplet codon in mammalian cells. Intriguingly, the trend of quadruplet codon decoding efficiency among the tested tRNA variants in mammalian cells was largely the same as that in E.coli. We subsequently demonstrate the utility of quadruplet codon decoding by the construction of the first HIV-1 mutant that lacks any in-frame amber nonsense codons and can be precisely activated by the decoding of a genomically embedded UAGA codon with an unnatural amino acid. Such conditionally activatable HIV-1 mutant can likely facilitate both fundamental investigations of HIV-1 as well as vaccine developments. The use of quadruplet codon, instead of an amber nonsense codon, to control HIV-1 replication has the advantage in that the correction of a frameshift caused by a quadruplet codon is much less likely than the reversion of an amber codon back into a sense codon in HIV-1.

Talk title Bridging the gap between RNA editing and modification: A 10‐year solution to a 25‐year problem

All types of nucleic acids in cells undergo naturally occurring chemical modifications, including DNA, rRNA, mRNA, snRNA, and most prominently tRNA. Over 100 different modifications have been described and every position in the purine and pyrimidine bases has been seen modified; the sugar is often also modified. Despite recent progress, the mechanism for the synthesis of most modifications is not fully understood partly due to difficulty in reconstituting modification enzyme activity in vitro. Cytosine to uridine (C to U) editing of tRNAs in eukaryotes was first discovered over 20 years ago, yet due to the lack of an in vitro assay, the editing enzyme is still at large and the editing mechanism remains unsolved. In this work we show that cytosine 32 in the anticodon loop of tRNAThr is methylated to 3‐methylcytosine (m3C) as a pre‐requisite for C to U deamination. Formation of m3C in vitro requires the presence of both TbTRM140 (the m3C methyltransferase) and the TbADAT2/3 (the A to I editing deaminase). Once m3C is formed the same set of enzymes are required for further deamination from m3C to form 3‐methyluridine (m3U). In our previous work, we also showed that TbADAT2/3 was a highly mutagenic enzyme randomly deaminating DNA. Now we provide evidence that when co‐expressed with the methylase its mutagenic ability is kept in check via complex formation with TRM140. This helps explain how T. brucei escapes “wholesale deamination” of its genome while harboring both enzymes in its nuclear compartment. This observation has implications for the control of another mutagenic deaminase, human AID, and provides a rationale for its regulation.Support or Funding InformationNIH GM084065This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Lack of 2'-O-methylation in the tRNA anticodon loop of two phylogenetically distant yeast species activates the general amino acid control pathway.

Modification defects in the tRNA anticodon loop often impair yeast growth and cause human disease. In the budding yeast Saccharomyces cerevisiae and the phylogenetically distant fission yeast Schizosaccharomyces pombe, trm7Δ mutants grow poorly due to lack of 2'-O-methylation of C32 and G34 in the tRNAPhe anticodon loop, and lesions in the human TRM7 homolog FTSJ1 cause non-syndromic X-linked intellectual disability (NSXLID). However, it is unclear why trm7Δ mutants grow poorly. We show here that despite the fact that S. cerevisiae trm7Δ mutants had no detectable tRNAPhe charging defect in rich media, the cells constitutively activated a robust general amino acid control (GAAC) response, acting through Gcn2, which senses uncharged tRNA. Consistent with reduced available charged tRNAPhe, the trm7Δ growth defect was suppressed by spontaneous mutations in phenylalanyl-tRNA synthetase (PheRS) or in the pol III negative regulator MAF1, and by overexpression of tRNAPhe, PheRS, or EF-1A; all of these also reduced GAAC activation. Genetic analysis also demonstrated that the trm7Δ growth defect was due to the constitutive robust GAAC activation as well as to the reduced available charged tRNAPhe. Robust GAAC activation was not observed with several other anticodon loop modification mutants. Analysis of S. pombe trm7 mutants led to similar observations. S. pombe Trm7 depletion also resulted in no observable tRNAPhe charging defect and a robust GAAC response, and suppressors mapped to PheRS and reduced GAAC activation. We speculate that GAAC activation is widely conserved in trm7 mutants in eukaryotes, including metazoans, and might play a role in FTSJ1-mediated NSXLID.

The complete mitochondrial DNA sequence from Kazachstania sinensis reveals a general +1C frameshift mechanism in CTGY codons.

The complete mitochondrial DNA (mtDNA) sequence from Kazachstania sinensis was analysed and compared to mtDNA from related yeasts. It contained the same set of genes; however, it only contained 23 tRNAs, as the trnR2 gene was absent. Most of the 12 introns within cox1, cob and rnl genes were inserted in the same sites as in other yeasts; however, two introns in rnl were in unusual positions. Traits such as gene order and GC cluster number were more related to Saccharomyces than to the other Kazachstania or linked clades. The most exceptional feature was the +1 frameshift in cox3, atp6 and cob open reading frames that was also found in other Kazachstania, Nakaseomyces delphensis and Candida glabrata. Comparison of DNA and protein sequences revealed the universal sites of +1C frameshifts were either CTGT or CTGC sequences. Moreover, an A→G substitution was found at position 37 in the anticodon stem loop tRNA gene for cysteine in all species with frameshifts but not in other sibling yeasts. This substitution allowed strong Watson-Crick base-pairing between an unmodified G (ACG) and the skipped C in the CTGY, leading to this quadruplet being read as cysteine.

Alternative Mode of E-Site tRNA Binding in the Presence of a Downstream mRNA Stem Loop at the Entrance Channel

Structured mRNAs positioned downstream of the ribosomal decoding center alter gene expression by slowing protein synthesis. Here, we solved the cryo-EM structure of the bacterial ribosome bound to an mRNA containing a 3' stem loop that regulates translation. Unexpectedly, the E-site tRNA adopts two distinct orientations. In the first structure, normal interactions with the 50S and 30S E site areobserved. However, in the second structure, although the E-site tRNA makes normal interactions with the 50S E site, its anticodon stem loop moves ∼54Å away from the 30S E site to interact with the 30S head domain and 50S uL5. This position of the E-site tRNA causes the uL1 stalk to adopt a more open conformation that likely represents an intermediate state during E-site tRNA dissociation. These results suggest that structured mRNAs at the entrance channel restrict 30S subunit movement required during translation to slow E-site tRNA dissociation.

Homologous VapC Toxins Inhibit Translation and Cell Growth by Sequence-Specific Cleavage of tRNAfMet.

Type II toxin-antitoxin (TA) systems play a critical role in the establishment and maintenance of bacterial dormancy. They are composed of a protein toxin and its cognate protein antitoxin. They function to regulate growth under conditions of stress, such as starvation or antibiotic treatment. As cellular proteases degrade the antitoxin, which normally binds and neutralizes the toxin, this frees the toxin to act on its cellular targets and arrest bacterial growth. TA systems are of particular concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae (NTHi), as dormancy may lead to chronic infections and failure of antibiotic treatment. Many targets of VapC toxins have not been identified, to date, and this knowledge is crucial to understanding how toxins control the establishment and maintenance of bacterial dormancy. Accordingly, we characterized the target specificity of the VapC toxins from the two paralogous NTHi vapBC TA systems. RNA sequencing and Northern blot analysis revealed that VapC1 and VapC2 cleave tRNAfMet in the anticodon loop. Overexpression of tRNAfMet suppresses VapC toxicity, suggesting that translation inhibition results from the depletion of tRNAfMet These experiments also identified base pairs in the tRNAfMet anticodon stem that play a key role in VapC-specific cleavage of the tRNA. Together these findings suggest the potential for NTHi VapC1 and VapC2 to induce dormancy by sequence-specific cleavage of tRNAfMetIMPORTANCE Bacterial persistence is a significant concern in regard to pathogenic organisms, such as nontypeable Haemophilus influenzae, as it can result in recurrent and chronic infections. Toxin-antitoxin systems can lead to persistence by causing bacteria to enter a slow-growing state that renders them antibiotic tolerant. Type II toxin components affect a wide variety of bacterial targets in order to elicit dormancy, and for many toxin-antitoxin systems, these mechanisms are not well understood. Thus, in order to understand how vapBC toxin-antitoxin systems cause dormancy, it is crucial to investigate the substrate specificity of VapC toxins. This study identifies the target of the VapC1 and VapC2 toxins from NTHi and takes important steps toward understanding the specificity of these toxins for their tRNA target.

Systematic Analysis of the Binding Surfaces between tRNAs and Their Respective Aminoacyl tRNA Synthetase Based on Structural and Evolutionary Data.

To determine the mechanism underlying the flow of genetic information, it is important to understand the relationship between a tRNA and its binding enzyme, a member of the aminoacyl-tRNA synthetase (aaRS) family. We have developed a novel method to project the interacting regions of tRNA–aaRS complexes, obtained from their three-dimensional structures, onto two-dimensional space. The interacting surface between each tRNA and its aaRS was successfully identified by determining these interactions with an atomic distance threshold of 3.3 Å. We analyzed their interactions, using 60 mainly bacterial and eukaryotic tRNA–aaRS complexes, and showed that the tRNA sequence regions that interacted most strongly with each aaRS are the anticodon loop and the CCA terminal region, followed by the D-stem. A sequence conservation analysis of the canonical tRNAs was conducted in 83 bacterial, 182 archaeal, and 150 eukaryotic species. Our results show that the three tRNA regions that interact with the aaRS and two additional loop regions (D-loop and TΨC-loop) known to be important for formation of the tRNA L-shaped structure are broadly conserved. We also found sequence conservations near the tRNA discriminator in the Bacteria and Archaea, and an enormous number of noncanonical tRNAs in the Eukaryotes. This is the first global view of tRNA evolution based on its structure and an unprecedented number of sequence data.

Glycyl-tRNA Synthetase as a Potential Universal Regulator of Translation Initiation at IRES-I

A full analysis has been conducted of the sequences and secondary structures of viral type-I or related IRESs identified in all of the elements that correspond to the previously described minimal fragment of the enterovirus C IRES, which mimics the glycine tRNA anticodon hairpin in the IRES structure and is necessary for the specific binding of glycyl-tRNA synthetase. Experiments on human glycyl-tRNA synthetase binding with the mRNA fragments of several taxonomically distant viruses showed that the binding constants of these complexes are similar. These results indicate that the regulation of translation initiation via glycyl-tRNA synthetase must be a universal mechanism for these viruses and the corresponding parts of their mRNAs must have similar spatial structures. Furthermore, at least one additional mRNA hairpin with the glycyl anticodon loop has been found in all analyzed viral type-I IRESs. It seems plausible that this extra hairpin is associated with the second RNA-binding site of the glycyl-tRNA synthetase dimer and stabilizes its complex with the viral mRNA.

Preferences of AAA/AAG codon recognition by modified nucleosides, τm5s2U34 and t6A37 present in tRNALys

Deficiency of 5-taurinomethyl-2-thiouridine, τm5s2U at the 34th ‘wobble’ position in tRNALys causes MERRF (Myoclonic Epilepsy with Ragged Red Fibers), a neuromuscular disease. This modified nucleoside of mt tRNALys, recognizes AAA/AAG codons during protein biosynthesis process. Its preference to identify cognate codons has not been studied at the atomic level. Hence, multiple MD simulations of various molecular models of anticodon stem loop (ASL) of mt tRNALys in presence and absence of τm5s2U34 and N6-threonylcarbamoyl adenosine (t6A37) along with AAA and AAG codons have been accomplished. Additional four MD simulations of multiple ASL mt tRNALys models in the context of ribosomal A-site residues have also been performed to investigate the role of A-site in recognition of AAA/AAG codons. MD simulation results show that, ASL models in presence of τm5s2U34 and t6A37 with codons AAA/AAG are more stable than the ASL lacking these modified bases. MD trajectories suggest that τm5s2U recognizes the codons initially by ‘wobble’ hydrogen bonding interactions, and then tRNALys might leave the explicit codon by a novel ‘single’ hydrogen bonding interaction in order to run the protein biosynthesis process smoothly. We propose this model as the ‘Foot-Step Model’ for codon recognition, in which the single hydrogen bond plays a crucial role. MD simulation results suggest that, tRNALys with τm5s2U and t6A recognizes AAA codon more preferably than AAG. Thus, these results reveal the consequences of τm5s2U and t6A in recognition of AAA/AAG codons in mitochondrial disease, MERRF.

Novel Genomic and Evolutionary Perspective of Cyanobacterial tRNAs.

Transfer RNA (tRNA) plays a central role in protein synthesis and acts as an adaptor molecule between an mRNA and an amino acid. A tRNA has an L-shaped clover leaf-like structure and contains an acceptor arm, D-arm, D-loop, anti-codon arm, anti-codon loop, variable loop, Ψ-arm and Ψ-loop. All of these arms and loops are important in protein translation. Here, we aimed to delineate the genomic architecture of these arms and loops in cyanobacterial tRNA. Studies from tRNA sequences from 61 cyanobacterial species showed that, except for few tRNAs (tRNAAsn, tRNALeu, tRNAGln, and tRNAMet), all contained a G nucleotide at the 1st position in the acceptor arm. tRNALeu and tRNAMet did not contain any conserved nucleotides at the 1st position whereas tRNAAsn and tRNAGln contained a conserved U1 nucleotide. In several tRNA families, the variable region also contained conserved nucleotides. Except for tRNAMet and tRNAGlu, all other tRNAs contained a conserved A nucleotide at the 1st position in the D-loop. The Ψ-loop contained a conserved U1-U2-C3-x-A5-x-U7 sequence, except for tRNAGly, tRNAAla, tRNAVal, tRNAPhe, tRNAThr, and tRNAGln in which the U7 nucleotide was not conserved. However, in tRNAAsp, the U7 nucleotide was substituted with a C7 nucleotide. Additionally, tRNAArg, tRNAGly, and tRNALys of cyanobacteria contained a group I intron within the anti-codon loop region. Maximum composite likelihood study on the transition/transversion of cyanobacterial tRNA revealed that the rate of transition was higher than the rate of transversion. An evolutionary tree was constructed to understand the evolution of cyanobacterial tRNA and analyses revealed that cyanobacterial tRNA may have evolved polyphyletically with high rate of gene loss.

Disclosing the functional changes of two genetic alterations in a patient with Chronic Progressive External Ophthalmoplegia: Report of the novel mtDNA m.7486G>A variant

Chronic Progressive External Ophthalmoplegia (CPEO) is characterized by ptosis and ophthalmoplegia and is usually caused by mitochondrial DNA (mtDNA) deletions or mt-tRNA mutations. The aim of the present work was to clarify the genetic defect in a patient presenting with CPEO and elucidate the underlying pathogenic mechanism. This 62-year-old female first developed ptosis of the right eye at the age of 12 and subsequently the left eye at 45 years, and was found to have external ophthalmoplegia at the age of 55 years. Histopathological abnormalities were detected in the patient's muscle, including ragged-red fibres, a mosaic pattern of COX-deficient muscle fibres and combined deficiency of respiratory chain complexes I and IV. Genetic investigation revealed the “common deletion” in the patient's muscle and fibroblasts. Moreover, a novel, heteroplasmic mt-tRNASer(UCN) variant (m.7486G>A) in the anticodon loop was detected in muscle homogenate (50%), fibroblasts (11%) and blood (4%). Single-fibre analysis showed segregation with COX-deficient fibres for both genetic alterations. Assembly defects of mtDNA-encoded complexes were demonstrated in fibroblasts. Functional analyses showed significant bioenergetic dysfunction, reduction in respiration rate and ATP production and mitochondrial depolarization. Multilamellar bodies were detected by electron microscopy, suggesting disturbance in autophagy. In conclusion, we report a CPEO patient with two possible genetic origins, both segregating with biochemical and histochemical defect. The “common mtDNA deletion” is the most likely cause, yet the potential pathogenic effect of a novel mt-tRNASer(UCN) variant cannot be fully excluded.

RNA cytosine methyltransferase Nsun3 regulates embryonic stem cell differentiation by promoting mitochondrial activity

Chemical modifications of RNA have been attracting increasing interest because of their impact on RNA fate and function. Therefore, the characterization of enzymes catalyzing such modifications is of great importance. The RNA cytosine methyltransferase NSUN3 was recently shown to generate 5-methylcytosine in the anticodon loop of mitochondrial tRNAMet. Further oxidation of this position is required for normal mitochondrial translation and function in human somatic cells. Because embryonic stem cells (ESCs) are less dependent on oxidative phosphorylation than somatic cells, we examined the effects of catalytic inactivation of Nsun3 on self-renewal and differentiation potential of murine ESCs. We demonstrate that Nsun3-mutant cells show strongly reduced mt-tRNAMet methylation and formylation as well as reduced mitochondrial translation and respiration. Despite the lower dependence of ESCs on mitochondrial activity, proliferation of mutant cells was reduced, while pluripotency marker gene expression was not affected. By contrast, ESC differentiation was skewed towards the meso- and endoderm lineages at the expense of neuroectoderm. Wnt3 was overexpressed in early differentiating mutant embryoid bodies and in ESCs, suggesting that impaired mitochondrial function disturbs normal differentiation programs by interfering with cellular signalling pathways. Interestingly, basal levels of reactive oxygen species (ROS) were not altered in ESCs, but Nsun3 inactivation attenuated induction of mitochondrial ROS upon stress, which may affect gene expression programs upon differentiation. Our findings not only characterize Nsun3 as an important regulator of stem cell fate but also provide a model system to study the still incompletely understood interplay of mitochondrial function with stem cell pluripotency and differentiation.

Characterization of redundant tRNAIles with CAU and UAU anticodons in Lactobacillus plantarum.

In most eubacteria, the minor AUA isoleucine codon is decoded by tRNAIle2, which has a lysidine (L) in the anticodon loop. The lysidine is introduced by tRNAIle-lysidine synthetase (TilS) through post-transcriptional modification of cytidine to yield an LAU anticodon. Some bacteria, Lactobacillus plantarum for example, possess two tRNAIle2(UAU) genes in addition to, two tRNAIle2(CAU) genes and the tilS gene. tRNA expression from all these genes would generate redundancy in a tRNA that decodes a rare AUA codon. In this study, we investigated the tRNA expression from these genes in L. plantarum and characterized the corresponding tRNAs. The tRNAIle2(CAU) gene products are modified by TilS to produce tRNAIle2(LAU), while tRNAIle2(UAU) lacks modification especially in the anticodon sequence. We found that tRNAIle2(LAU) is charged with isoleucine but tRNAIle2(UAU) is not. Our results suggest that the tRNAIle2 redundancy may be related to different roles of these tRNAs in the cell.

Conformational preferences and structural analysis of hypermodified nucleoside, peroxywybutosine (o2yW) found at 37th position in anticodon loop of tRNAPhe and its role in modulating UUC codon-anticodon interactions

Hypermodified bases present at 3′-adjacent (37th) position in anticodon loop of tRNAPhe are well known for their contribution in modulating codon-anticodon interactions. Peroxywybutosine (o2yW), a wyosine family member, is one of such tricyclic modified bases observed at the 37th position in tRNAPhe. Conformational preferences and three-dimensional structural analysis of peroxywybutosine have not been investigated in detail at atomic level. Hence, in the present study quantum chemical semi-empirical RM1 and multiple molecular dynamics (MD) simulations have been used to study structural significance of peroxywybutosine in tRNAPhe. Full geometry optimizations over the peroxywybutosine base have also been performed using ab-initio HF-SCF (6-31G**), DFT (B3LYP/6-31G**) and semi-empirical PM6 method to compare the salient properties. RM1 predicted most stable structure shows that the amino-carboxy-propyl side chain of o2yW remains ‘distal’ to the five membered imidazole ring of tricyclic guanosine. MD simulation trajectory of the isolated peroxy base showed restricted periodical fluctuations of peroxywybutosine side chain which might be helpful to maintain proper anticodon loop structure and mRNA reading frame during protein biosynthesis process. Another comparative MD simulation study of the anticodon stem loop with codon UUC showed various properties, which justify the functional implications of peroxywybutosine at 37th position along with other modified bases present in ASL of tRNAPhe. Thus, this study presents an atomic view into the structural properties of peroxywybutosine, which can be useful to determine its role in the anticodon stem loop in context of codon-anticodon interactions and frame shift mutations.

The complete mitochondrial genome of Anoplocnemis curvipes F. (Coreinea, Coreidae, Heteroptera), a pest of fresh cowpea pods

The complete 16,345-bp mitochondrial genome of the agriculturally destructive pod sucking pest, the giant coreid bug, Anoplocnemis curvipes (Hemiptera: Coreidae), was assembled from paired-end Illumina HiSeq 2500 reads. The A. curvipes mitochondrial genome consists of 13 protein coding genes (PCGs), 22 tRNAs, 2 rRNAs and a control region in the order and orientation typical among insects. PCG initiation codons (ATG, ATC, ATT and ATA) with termination codon (TAA) are used with the exception of TAG stop codons by Cytb and ND3. All tRNA genes fold into predicted cloverleaf secondary structures having requisite triplets on the anticodon loop, apart from tRNA-Ser1 (AGN) whose dihydrouridine (DHU) arm forms a simple loop. The phylogenetic analysis of hemipteran mitogenomes clusters to the family level and supports the monophyly of the five superfamilies in Pentatomomorpha of Hemiptera. The Coreoidea and Pyrrhocoroidea are sister groups, while Coreidae and Alydidae are sister groups to Rhopalidae. These analyses provide insight to mitogenomics and evolutionary relationships among pentatomoid insects.

Protein-tRNA Agarose Gel Retardation Assays for the Analysis of the <em>N</em><sup>6</sup>-threonylcarbamoyladenosine TcdA Function

We demonstrate methods for the expression and purification of tRNA(UUU) in Escherichia coli and the analysis by gel retardation assays of the binding of tRNA(UUU) to TcdA, an N6-threonylcarbamoyladenosine (t6A) dehydratase, which cyclizes the threonylcarbamoyl side chain attached to A37 in the anticodon stem loop (ASL) of tRNAs to cyclic t6A (ct6A). Transcription of the synthetic gene encoding tRNA(UUU) is induced in E. coli with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells containing tRNA are harvested 24 h post-induction. The RNA fraction is purified using the acid phenol extraction method. Pure tRNA is obtained by a gel filtration chromatography that efficiently separates the small-sized tRNA molecules from larger intact or fragmented nucleic acids. To analyze TcdA binding to tRNA(UUU), TcdA is mixed with tRNA(UUU) and separated on a native agarose gel at 4 °C. The free tRNA(UUU) migrates faster, while the TcdA-tRNA(UUU) complexes undergo a mobility retardation that can be observed upon staining of the gel. We demonstrate that TcdA is a tRNA(UUU)-binding enzyme. This gel retardation assay can be used to study TcdA mutants and the effects of additives and other proteins on binding.